The ultimate objective of this project is to understand the role of glucocorticoid binding and the physiology of hormone action. Toward this end, we have studied several aspects of glucocorticoid receptor physiology, including the kinetics of receptor binding, activation and nuclear translocation. Progress has been made on three objectives of this project: isolation of ACTH mRNA and production of ACTH cDNA, analysis of in vivo receptor activation and nuclear binding of the glucocorticoid receptor and characterization of the glucocorticoid receptor itself. We have characterized approximately 1300 nucleotide cDNA to ACTH mRNA cloned into pBR322. This clone has been sequenced, used to identify ACTH mRNA and polysomal RNA extracted from AtT-20 cells and used by another laboratory at Vanderbilt to identify and characterize ACTH mRNA from ectopic hormone-producing tumors of humans. The extent of in vivo activation and nuclear translocation produced in AtT-20 cells by four glucocorticoid agonists has been determined. This marks the first time that receptor activation and nuclear translocation have been correlated in vivo. It was found that for dexamethasone, triamcinolone acetonide, prednisolone and corticosterone there was a rigid correlation between binding affinity, activation and nuclear binding. Furthermore, although the extent of activation varied with each steroid, the fraction of activated receptor which was translocated was similar (approximately 60%) in each case, lending support to the concept that nuclear translocation is a simple partitioning phenomenon. Last, the rat glucocorticoid receptor has been purified and monoclonal antibodies have been produced to it which also recognize the mouse glucocorticoid receptor. These reagents will make further analysis of the mouse glucocorticoid receptor possible. As of this writing, stable hybridomas producing monoclonal antibodies to the glucocorticoid receptor have not been described. Specific objectives for the coming year are: (1) to isolate the mouse glucocorticoid receptor gene, using a monoclonal antibody to the receptor developed in this laboratory; (2) to characterize the relationship between intact cell uptake and biological potency of steroids of various potency; (3) to purify the mouse and rat glucocorticoid receptor with an immunoaffinity column.